Glyphosate is a widely used component in herbicides. Glyphosate inhibits 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase, or EPSPS), which is involved in the synthesis of aromatic amino acids in plant cells. Inhibition of EPSPS effectively disrupts protein synthesis and thereby kills the affected plant cells. Because glyphosate is non-selective, it kills both weeds and crop plants. Accordingly, there is a need to produce transgenic crop plants that are resistant to glyphosate.
Recombinant DNA technology has been used to create mutant EPSP synthases that are glyphosate-resistant. These mutant EPSP synthases can be transformed into plants and confer glyphosate-resistance upon the transformed plants. Examples of mutant EPSP synthases and glyphosate-resistant transgenic plants are illustrated in U.S. Pat. Nos. 6,040,497 and 5,554,798, 5,310,667 and WO 00/66748.
Current plant transformation technology employs chimeric expression vectors. These vectors include regulatory sequences, such as enhancers or promoters, that are heterologous to the EPSPS genes. For instance, WO 00/66748 fuses enhancers from CaMV 35S, FMV 35S, rice actin 1, rice GOS2, maize polyubiquitin, or barley plastocyanin genes to a glyphosate-resistant EPSPS coding sequence in order to enhance the expression of the glyphosate-resistant EPSPS in transformed plant cells.
No one has used a complete expression cassette of the EPSP synthase gene isolated from the genome of a donor plant and mutated to give glyphosate resistance. In one embodiment of the present invention, the expression cassette of the EPSP synthase gene consists of a native EPSPS 5′ regulator sequence, a coding sequence (with or without introns) encoding a glyphosate-resistant EPSPS which includes a native transit peptide, and a native EPSPS 3′ regulatory sequence (such as an EPSPS transcriptional terminator). The fact that such an expression cassette is sufficient to provide glyphosate resistance is surprising. Moreover, the use of the native EPSPS 5′ and/or 3′ regulatory sequences simplifies the process of constructing expression vectors suitable for plant transformation.
Suitable sources of EPSP synthase genes include dicotyledonous plants, such as Arabidopsis thaliana, and mono-cotyledonous plants, such as Zea mays, Arabidopsis thaliana has two EPSP synthase genes (epm1 and epm2). The present invention includes use of one or both of mutated epm1 and epm2 to confer resistance to glyphosate. Mutated EPSP synthase genes from Zea mays or other plants can also be used for transforming plant cells to make glyphosate-resistant plants.